Determination of glucose blood levels is one of the most frequently performed analyses in hospital laboratories. Glucose determinations are utilized for diagnosis and tracking of carbohydrate metabolism abnormalities such as hypoglycemia, diabetes mellitus as well as other conditions such as glucose levels in cerebrospinal fluid where meningitis is suspected.
Blood glucose levels fluctuate within a fairly narrow range. Variation in blood glucose is generally a result of changes in gross dietary circumstances such as feeding versus periods of fasting. However, detection of blood glucose levels which are above or below the normal range are indicative of some type of disease state. The most common disease associated with high blood glucose levels is diabetes mellitus. Serious diseases are rarely associated with low blood glucose levels and the common cause of low glucose blood levels is that the blood sample was taken during a fasting state in the subject.
In the insurance industry the health of the candidate is evaluated at the time of application for the insurance contract. It is common practice for an insurance company to send an on-site examiner to the individual seeking insurance and have a body fluid sample--blood--collected at a location most convenient for the individual. Heretofore, on-site examiners have collected blood by conventional methods such as venipuncture which results in large sample volumes. Recently an alternative method, by pricking the finger of the subject and dropping blood onto an sorbent layer, has become favored. The latter technique requires a much smaller blood volume and provides sample which ma be conveniently shipped to a central laboratory for testing.
"On-site" sample collection is by definition the collection of a sample under non-laboratory controlled conditions. One can either test the sample promptly on-site, which while possible is neither economical nor acceptable to insurance companies ordering such tests, or a valid test can be conducted by a properly qualified technician in a certified laboratory. Therefore, even though the sample maybe collected by a non-professional on-site, it must be conveyed to a central laboratory.
In many cases it is convenient, as well as necessary, to ship the sample to the laboratory by mail or courier in order to minimize the number of operations at the laboratory facility. In such case it is beneficial if the liquid sample can be reduced to a solid phase so as to avoid shipment of fluid blood samples. As a result it has recently been found advantageous to ship body fluid samples, blood in particular, as a dried spot on a sorbent matrix. In this manner the sample volume is reduced and the sample weight commensurately reduced by evaporation of the fluid component. The use of dried blood spot samples also avoids separation of plasma or serum from the cells by centrifugation. Thus is eliminated the inconvenience of either carrying about the centrifuge or taking samples to a central location for centrifugation within a short time after drawing the blood sample.
The technique for obtaining such a dried blood spot on a sorbent matrix involves pricking the finger of the subject and dropping the blood onto the sorbent layer at a specifically designated location and saturating that location with blood. The saturation of the sorbent matrix is followed by drying of the blood spot for sometime prior to insertion of the sorbent matrix into a mailing envelope for shipment to the laboratory.
For many components of blood the handling of the sample after application to the sorbent paper or matrix and the time of drying is not a critical factor for subsequent analysis. However, in the case of blood glucose determinations the handling of the blood sample by the on-site examiner is a critical aspect of the ultimate accuracy of glucose measurement in the sample. Once the blood sample is removed from the subject the physiological functioning of the cells within the blood sample continues. In the case of glucose, the entirety of the Embden-Meyerhof pathway continues to operate for sometime within the cells. This pathway is the principal method by which carbohydrates, and glucose in particular, are metabolized. Therefore, even though the blood sample is removed from the body the concentration of glucose within the blood sample will decrease over time unless glycolysis within the Embden-Meyerhof pathway is inhibited.
In blood samples in which glycolysis is not inhibited, the loss of glucose from the sample is generally estimated at 10 mg/deciliter(dl)/hour. Thus it is necessary to provide some type of means to inhibit glycolysis within the Embden-Meyerhof pathway in situations where immediate analysis of the glucose levels within a blood sample is not possible. When it is appreciated that the normal concentration of glucose in the blood of an adult is approximately 70-105 mg/dl of serum, it is apparent that the passage of even short periods of time can cause substantial reduction in the glucose content of the sample. Clearly such a high rate of glucose loss in a blood sample can rapidly cause incorrect measurement of glucose concentration in a subject's blood when applied to a untreated sorbent matrix.
Such a decrease in glucose concentration will be found when a blood sample is applied to a sorbent layer for shipment to a laboratory for subsequent glucose concentration analysis. One method of inhibiting the continuation of glycolysis in a blood sample applied to a sorbent layer is drying of the blood sample. The drying of the blood sample on the sorbent causes cessation of cellular activity by dehydration and subsequent termination of glycolysis within the cell. For the analysis of the glucose concentration in a blood sample to be accurate it is necessary that the drying of the sample on the sorbent layer be conducted immediately and that the blood sample be thoroughly dried prior to shipment of the sample to the laboratory.
However, in the course of sample collection by on-site examiners it is a frequent occurrence that the drying of the sample is delayed or that drying of the sample is never completely accomplished. This variation in the drying of the sample leads to variation in the decrease of glucose in samples taken from various subjects as well as in samples taken from the same subject.
In normal on-site sampling the on-site examiner is to obtain the blood sample by lancing a finger of the subject and squeezing drops of blood onto a sorbent layer. The application of the blood to the absorbent layer is to be immediately followed by drying the samples rapidly as possible. This is often accomplished by use of a heat gun to speed the drying process, but heating can also lead to baking the blood onto the sorbent matrix from which it may not then be eluted and therefore be unsuitable for analysis. When drying is accomplished quickly and rapidly minimum glucose loss due to cellular glycolysis is observed.
Since on-site examiners are not professional medical technicians, it is not uncommon for deviations from the approved protocol to occur. The drying of samples may not be done at all or very poorly or for variable time intervals or various portions of a single sample will be subjected to different rates of drying. This will result in disparate results within the same subject as well as between similar subjects. Also, it is not uncommon for samples to be placed in a shipping package without having been dried at all. Once blood is absorbed into the sorbent matrix, the examiner may assume that proper application is done and it is time to ship the sample. In such a case cellular functioning may continue for sometime and substantially deplete the glucose concentration in the sample rendering the sample unreliable for glucose determination.
Therefore, it is an object of the present invention to provide a method of glucose determination in a blood sample which will avoid the difficulties presented by the prior art methods.
Another object of the present invention is to provide a method of glucose determination which is independent of sample handling.
Yet another object of the present invention is to provide a method of glucose determination from a dried blood spot which avoids the variations in glucose concentration caused by improper and variable drying of the sample.
Yet another object of the present invention is to provide a method of field or on-site blood collection for subsequent glucose determination which rapidly and conveniently stabilizes the glucose content of the blood sample.
Another object of the present invention is to provide a method of field or on-site blood sampling for subsequent glucose determination which rapidly inhibits glycolysis in the blood sample.
Yet another object of the present invention is to provide a method of stabilizing a blood sample for subsequent glucose determination through collection of the blood sample on a conveniently and easily shipped substrate.
Yet another object of the present invention is to provide a method of obtaining a blood sample for subsequent glucose determination which involves drying the blood sample while maintaining a glucose concentration near that of a freshly obtained blood sample.
Yet another object of the present invention is to provide a method for inhibiting glucose degradation in a field blood sample which can be utilized at low cost.
Yet another object of the present invention is to provide a method which assists in avoiding the need to take repeat samples from subjects due to improper sampling procedures.
Other objects and advantages of this invention will become apparent from the following detailed description wherein is set forth by way of illustration and example, an embodiment of this invention.